UNIVERSITI PUTRA MALAYSIA

FACTORS AFFECTING THE OPTIMIZATION OF LIPASE-CATALYSED PALM-BASED ESTERS SYNTHESIS

ERIN RYANTIN GUNAWAN.

FS 2005 35

FACTORS AFFECTING THE OPTIMIZATION OF LIPASE- CATALYSED PALM-BASED ESTERS SYNTHESIS

ERIN RYANTIN GUNAWAN

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfillment of the Requirement

for the Degree of Doctor of Philosophy

DEDICATION

I dedkate this thesis to allmy farnib andI6u Pertiwi.. . . . . . . ..Indonesia

Abstract of thesis presented to the Senate of Universiti h t r a Malaysia in fulfillment of the requirement for the degree of Doctor

of Philosophy

FACTORS AFFECTING THE OPTIMIZATION OF LIPASE CATALYSED PALM-BASED ESTERS SYNTHESIS

ERIN RYANTIN GUNAWAN

July 2005

Chairman: Professor Mahiran Basri, PhD

Faculty: Science

The lipase synthesis of esters using palm oil fractions and long

chain alcohol as substrates was carried out. Various lipases were

tested for their suitability for the reaction. Among the enzymes

tested, Lipozyme IM exhibited the highest percentage yield of palm

oil ester (more than 75%) compared with the other lipases. Two

methods, classical method (one-variable at-a-time) and response

surface methodology (RSM), were employed for optimization of the

reaction.

By the classical method, five parameters such as reaction time,

temperature, amount of enzyme, molar ratio of substrates and

various organic solvents of the reaction system were investigated.

The optimum yield was achieved at the reaction temperature of 40

- 500C for palm oil (PO) and 400C for palm kernel oil (PKO)

alcoholysis, a reaction time of 5 - 7 h for PO and 7 - 10 h for PKO,

0.15 g of enzyme for both PO and PKO alcoholysis, molar ratio at 3:

1 (alcohol: PO or PKO), and the best solvent for the reactions was

hexane. Percentage yields of esters obtained at these optimum

reaction conditions were 83, 80 and 81% for refined, bleached and

deodorized (RBD) palm oil, RBD palm stearin (PS) and RBD palm

olein (PL), respectively and 87, 90 and 86% for RBD palm kernel

oil, RBD palm kernel stearin (PKS) and RBD palm kernel olein

(PKL) , respectively.

The classical method of optimization involves varying one

parameter a t a time and keeping the other parameters constant.

However, this method is inefficient as it fails to understand

relationships between the variables (reaction time, temperature,

molar ratio and amount of enzyme) and the response (percentage

yield). RSM is an effective statistical technique for the investigation

of complex processes. RSM comprising of a five-level, four-factor

central composite rotatable design (CCRD) was used to evaluate

the interactive effects of synthesis reaction time (2.5 - 10 h of PO, 5

- 15 h of PKO), temperature (30 - 700C of PO, 30 - 500C of PKO),

amount of enzyme (0.1 - 0.2 g of PO or PKO) and substrates molar

ratio (1: 1 - 5: 1 alcohol to PO or PKO) on the percentage yield of

esters and to obtain the optimum conditions for enzyme-catalyzed

alcoholysis of palm-based ester.

The optimum conditions derived by RSM of PO and PKO were:

reaction time at 7.38 and 10 h, temperature of 53.9% and 44.20C,

amount of enzyme of 0.149 and 0.157g, and substrates molar ratio

3.41: 1 and 3.78: 1 (alcohol: PO or PKO), respectively. The actual

experimental yield of PO and PKO were 84.6% and 90.8% under

these optimum conditions, which compared well with the

maximum predicted value of 85.4% and 91.8% for PO and PKO,

respectively.

The composition of esters synthesized from PO at optimum

reaction condition are 0.8% of oleyl laurate, 3.8% of oleyl

myristate, 35.5% of oleyl palmitate, 4.5% of oleyl stearate, 33.3%

oleyl oleate and 6.2% of oleyl linoleate. Meanwhile, The

composition of esters synthesized from PKO are 0.7% of oleyl

caproate, 5.7% of oleyl caprylate, 3.7% of oleyl caprate, 36.7% of

laurate, 13.10% of oleyl myristate, 8.8% of oleyl palmitate, 2.5%

oleyl stearate, 14.1% of oleyl oleate and 2.4% of oleyl linoleate.

These optimum conditions were also used in alcoholysis of PS, PL,

PKS and PKL, which gave the average percentage yield of more

than 80%.

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia bagi memenuhi keperluan untuk ijazah Doktor Falsafah

FAKTOR-FAKTOR MEMPENGARUHI PENGOPTIMUMAN SINTESIS ESTER BERASASKAN KELAPA SAWIT

DIMANGKINKAN OLEH LIPASE

Oleh

ERIN RYANTIN GUNAWAN

Julai 2005

Pengerusi: Profesor Mahiran Basri, PhD

Fakulti: Sains

Sintesis ester lilin mengunakan pecahan minyak kelapa sawit and

alkohol rantai panjang sebagai substrat yang dimangkinkan oleh

lipase telah dijalankan. Dua kaedah iaitu kaedah klasik (satu-

variasi pada satu-masa) dan kaedah permukaan respon, telah

digunakan untuk mencari keadaan optima. Pelbagai jenis lipase

telah diuji kesesuaiannya untuk tindak balas. Di antara enzim

yang telah diuji, Lipozyrne IM memberi hasil peratusan ester lilin

kelapa sawit paling tinggi (lebih dari 75 %) berbanding lipase lain

yang diuji.

Dalarn kaedah klasik, lirna parameter iaitu masa tindak-balas,

suhu, jumlah enzim, nisbah molar substrat dan kepelbagaian

pelarut organik telah di kajiselidik.

Hasil optima telah dicapai pada suhu tindak balas alkoholisis 40-

50°C bagi minyak kelapa sawit (PO) dan 40°C bagi minyak isirung

kelapa sawit (PKO), masa tindak balas pada 5 - 7 jam bagi PO dan

pada 7 - 10 jam bagi PKO, jumlah enzim bagi 0.15 g untuk

alkoholisis PO dan PKO, nisbah molar substrat pada 3: 1 (alcohol:

PO atau PKO) dan pelarut terbaik bagi tindak balas adalah

heksana. Hasil peratusan ester lilin yang diperolehi mengunakan

keadaan optima ini adalah 83, 80 dan 81% bagi minyak kelapa

sawit yang dibersih, diluntur dan dinyahbau (RBD), RBD kelapa

sawit sterin (PS) d m RDB kelapa sawit o h , masing-masing 87, 90

dan 86% bagi RBD minyak kelapa sawit (PKL), RBD kelapa sawit

isirung sterin (PKS) dan RBD kelapa sawit isirung olin (PKL).

Kaedah klasik bagi pengoptimaan tennasuk memvariasikan satu

parameter pada satu masa dan mengekalkan selainnya boleh

digunakan. Walaubagaimanapun, kaedah ini tidak mencukupi

kerana ia gagal untuk memahami hubungkait di antara variable

(masa tindak balas, suhu, nisbah molar dan jumlah enzim) dan

respon (peratusan hasil). RSM adalah teknik statistik untuk

menyelidik proses yang komplek. RSM mengandungi lima-

peringkat, empat-faktor jenis pusat komposit putaran (CCRD)

telah digunakan untuk mengkaji kesan interaktif dalam sintesis

bagi masa tindak balas (2.5 - 10h bagi PO, 5 - 15 jam bagi PKO),

suhu (30 - 70°C bagi PO, 30 - 50°C bagi PKO), jumlah enzim (0.1 -

0.2 g bagi PO atau PKO) dan nisbah molar susbstrat ( 1: 1 -5: 1

alkohol bagi PO atau PKO) peratusan hasil ester lilin dan untuk

mendapatkan keadaan optima untuk alkoholisis bermangkinkan

enzim yang menghasilkan ester lilin berasaskan kelapa sawit.

Keadaan optima diperolehi melalui RSM bagi PO dan PKO adalah;

masa tindak balas pada 7.38 dan 10 jam, suhu pada 53.9 dan

44.Z°C, jumlah enzim pada 0.179 dan 0.157 g dan nisbah molar

substrat 3.41: 1 dan 3.78: 1 (alkohol: PO atau PKO), masing-

masing. Hasil sebenar dari eksperimen bagi PO dan PKO adalah

84.6% dan 90.8% di bawah keadaan optima, di mana ia sesuai

dengan nilai yang dianggar iaitu 85.4% dan 9 1.8% bagi PO dan

PKO. Keadaan optimum ini juga digunakan pada alkoholisis PS,

PL, PKS dan PKL, di mana ia telah memberikan purata hasil

peratusan lebih daripada 80%.

. . . Vlll

ACKNOWLEDGEMENTS

In The Name of ALLAH, The Most Merciful and Most Beneficent

All praises do to Allah, Lord of the universe. Only by His grace and

mercy this thesis can be completed.

This work was carried out with a hope to contribute towards the

expansion of our currently limited knowledge on Oleochemistry.

The completion of this thesis would have been impossible if not for

the assistance and direct involvement of so many kindhearted

individuals. Thus, I am very much indebted to my previous

mentors and I have no way of repaying such a dept except to

express my sincerest gratitude.

First and foremost, I am very grateful to my adviser Professor Dr.

Mahiran Basri, for her strong support, guidance, and patience for

the very enriching and thought provoking discussions and lectures

which helped to shape the thesis. She was always there to provide

everything I needed in the laboratory. I would also like to thank her

besides Prof. Dr. Abu Bakar Salleh for providing financial support

during the period of study through the IRPA research fund.

I am also grateful to Prof. Dr. Abu Bakar Salleh, Assoc. Prof. Dr.

Raja Noor Zaliha Abd. Rahman and Assoc. Prof. Dr. Mohd.

Basyaruddin Abd. Rahman in their capacities as members of

Supervisory Committee. Thank you for the comments and

suggestion, which contributed a lot towards the improvement of

the final manuscript. I am also indebted to the staff of the

Department of Chemistry, Universiti FWra Malaysia for their help

and cooperation.

Special thanks are extended to Dean of Fakultas Keguruan dan

Ilmu Pendidikan (FKIP), Rector of Universitas Mataram and

Direktorat Pendidikan Tinggi Departemen Pendidikan Nasional

Indonesia, which were allowed me to study in PhD level. Special

thanks are also extended to other 401 lab members (Dr. Siti

Salhah, Salina, Ita, Pei Sin, Azmahani, etc) who helped me in every

way possible and providing a congenial and enthusiastic

atmosphere in the laboratory. Acknowledgement is also extended

to Indonesian Student Association (PPI-UPM) that joined us in

sweet friendship and made life easier during my stay in Malaysia.

I wish to express my deepest gratitude to my parents, brothers and

sisters for their prayers, continuous moral support and unending

encouragement. Last but not least, I wish especially to

acknowledge my beloved husband, Dr. Dedy Suhendra, and my

dearest children Erdy Izzatuffikri, Erdanisa Aghnia Ilmani and

Erdy Muhammad Fakhri for their love, support, patience and

understanding.

I cer* that an Examination Committee met on 18th July 2005 to conduct the final examination of Erin Ryantin Gunawan on her Doctor of Philosophy thesis entitled "Factors AffecQng the Optimization of Lipase-Catalysed Palm-Based Esters Synthesis" in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The Committee recommends that the candidate be awarded the relevant degree. Members of the Examination Committee are as follows:

Zulkarnain Zainal, PhD Professor Faculty of Science Universiti Putra Malaysia (Chairman)

Dzulkefly Kuang Abdullah, PhD Professor Faculty of Science Universiti Putra Malaysia (Internal Examiner)

Nor Aripin Shamaan, PhD Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Internal Examiner)

Noorsaadah Abdul Rahman, PhD Professor Faculty of Science Universiti Malaya (External Examiner)

Date: 2 2 AUG 2005

xii

This thesis submitted to the Senate of Universiti Putra Malaysia and has been accepted a s fulfillment of the requirement for the degree of Doctor of Philosophy. The members of the Supervisory Committee are a s follows:

Mahiran Basri, PhD Professor Faculty of Science Universiti Putra Malaysia (Chairman)

Abu Bakar Salleh, PhD Professor Faculty of Biotechnology and Biomolecular Science Universiti Putra Malaysia (Member)

Raja Noor Zaliha Raja Abdul Rahman, PhD Associate Professor Faculty of Biotechnology and Biomolecular Science Universiti Putra Malaysia (Member)

Mohd. Basyaruddin Abdul Rahman, PhD Associate Professor Faculty of Science Universiti Putra Malaysia (Member)

AINI IDERIS, PhD Professor/ Dean School of Graduate Studies Universiti Putra Malaysia

Date: 0 8 SEP 2005

. . . Xl l l

DECLARATION

I hereby declare that the thesis is based on my original work except for quotations and citations, which have been duly acknowledged. I also declare that it has not been previously or concurrently submitted for any other degree at UPM or other institutions.

ERIN RYANTIN GUNAWAN

DATE: > o / ~ / w 05

xiv

TABLEOFCONTENTS

Page

DEDICATION ABSTRACT ABSTRAK ACKNOWLEDGEMENTS APPROVAL DECLARATION LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS

CHAPTER

INTRODUCTION

LITERATURE REVIEW

. . 11 ... 111

vi ix xii xiv xix xxi xxvi

Wax Ester 6 Properties of Wax Ester 6 Application of Wax Ester 8

Synthesis of Wax Ester 11 Chemical Synthesis 11 Enzymatic Synthesis 12 Analysis of Wax ester 14

Palm Oil 16 Chemistry of Palm Oil 17 Oleochemicals 21 Palm and Palm Kernel Oils as Raw 21 Material for Oleochemicals

Lipase as Biocatalyst Lipases in Oleochemical Industry

Optimization Studies Response Surface Methodology (RSM)

Factorial Design Central Composite Rotatable Design The Four-step Procedure in RSM

Application of RSM

MATERIALS AND METHODS

Materials and Equipments Methodology Preparation of Ester Standards

Esterification Reaction Isolation and Purification of Ester Standards

Synthesis of Esters Alcoholysis Reaction Product Isolation and Purification

Product Identification Thin Layer Chromatography (TLC) Gas Chromatography (GC) GC-Mass Spectroscopy Analysis Fourier Transform Infrared (FTIR) Spectroscopy

Screening of Lipase Optimization Studies

Effect of Reaction Time Effect of Reaction Temperature Effect of Amount of Enzyme Effect of Molar Ratio of Substrate Effect of Various Organic Solvents

Kinetic Study Alcoholysis Reaction a t Optimum Condition (Classical Method) Study on Interactive Effects of Enzymatic Reaction Parameters and Their Optimization Using RSM

Experimental Design Statistical and Graphical Analyses Optimization of Reaction and Model Validation Alcoholysis Reaction a t Optimum Condition (RSM)

Enzyme Reusability The Physical Properties Tests of The Products

Determination of Saponification Value (SV) Determination of Iodine Value Determination of Acid Value Solubility Test

xvi

RESULTS AND DISCUSSION

Synthesis of Ester Identification of The Products

Thin Layer Chromatography (TLC) FTIR Spectroscopy Gas Chromatography (GC) Analysis

Screening of Enzymes Optimization Studies of Palm Oil and Palm Kernel Oil Alcoholysis

Effect of Reaction Time Effect of Reaction Temperature Effect of Amount of Lipase Effect of Molar Ratio Effect of Organic Solvent Alcoholysis Reaction a t Optimum Conditions Ester Composition of The Products

Kinetic Study Response Surface Methodology (RSM)

Experimental Design Fitting Model and ANOVA Regression Analysis

Parameter Effects Interactive Effect of Reaction Time and Temperature Interactive Effect of Substrate Molar Ratio and Temperature Interactive Effect of Varying Amount of Enzyme and Temperature Interactive Effect of Reaction Time and Substrate Molar Ratio Interactive Effect of Reaction Time and Amount of Enzyme Interactive Effect of Varying Amount of Enzyme and Substrate Molar Ratio

Contour Plots Optimization of Reaction by RSM

Alcoholysis Reactions a t Optimum Conditions Optimized by RSM Composition of Esters from Palm Oil, Olein and Stearin Ester Optimized by RSM Comparison of One-variable-at-time (Conventional) Method and RSM on Percentage Yield of Esters

Operational Stability Physical Properties of Ester

Saponification Value and Molecular Weight Iodine Value (IV) Acid Value (AV) Solubility of The Products Viscosity of The Products

CONCLUSIONS

Recommendation for Further Studies

BIBLIOGRAPHY

APPENDICES

BIODATA OF THE AUTHOR

xviii

LIST OF TABLES

Table

Physical and chemical properties of palm oil and palm kernel olein

Fatty acid compositions (96) in palm oils

Fatty acid compositions (%) in palm kernel oils

Lipases and their sources

Coded and Actual Level Combination for a Five- level, Four-Variable Fractional Factorial Design of Palm Oil alcoholysis

Coded and Actual Level Combination for a Five- level, Four-Variable Fractional Factorial Design of Palm Kernel Oil alcoholysis

Reactions mixture under optimized condition

Composition of esters synthesized from palm oil fractions at the optimum condition of palm oil alcoholysis

Composition of esters synthesized from palm kernel oil fractions at the optimum condition of palm kernel oil alcoholysis

Kinetic and thermodynamic parameters for the alcoholysis of palm oil/palm kernel oil and oleyl alcohol

Central composite rotatable quadratic polynomial model, experimental data, actual and predicted values for 5-level-4-factor response surface analysis of palm oil

Page

18

xix

Central composite rotatable quadratic polynomial model, experimental data, actual and predicted values for 5-level-4-factor response surface analysis of palm kernel oil

ANOVA for joint test of palm oil

ANOVA for joint test of palm kernel oil

Values and Significance of Regression Coefficients of Coded Factors for Palm Oil Alcoholysis.

Values and Significance of Regression Coefficients of Coded Factors for Palm Kernel Oil Alcoholysis

Solutions of optimum conditions of palm oil

Solutions of optimum conditions of palm kernel oil

Composition of ester from palm oil fraction alcoholysis a t optimum condition optimized by RSM

Composition of ester from palm kernel oil fraction alcoholysis at optimum condition optimized by RSM

The saponification value of esters

The saponification value of esters from different commercial sources

The iodine values of esters

The iodine values of commercial esters

The acid values of esters

The acid values of commercial esters

Viscosity of esters

Viscosity of commercial esters

LIST OF FIGURES

Figure

1

Page

63 Thin layer chromatography of standard ester catalyzed by Lipozyme

FTIR spectrum

Chromatogram of ester standards

Chromatogram of esters from alcoholysis of palm oil

Chromatogram of esters from alcoholysis of palm kernel olein

Screening of lipase

Effect of reaction time on the total yield of palm oil and palm kernel oil alcoholysis

Effect of reaction time on palm oil alcoholysis

Effect of reaction time on palm kernel oil alcoholysis

Effect of temperature on the total yield of palm oil and palm kernel oil alcoholysis

Effect of temperature on palm oil alcoholysis

Effect of temperature on palm kernel oil alcoholysis

Effect of amount of lipase on the total yield of palm oil and palm kernel oil alcoholysis

Effect of amount of lipase on palm oil alcoholysis

Effect of amount of lipase on palm kernel oil alcoholysis

xxi

Effect of molar ratio of substrate on the total yield of palm oil and palm kernel oil alcoholysis

Effect of molar ratio of substrate on palm oil alcoholysis

Effect of molar ratio of substrate on palm kernel oil alcoholysis

Effect of various organic solvents on the total yield of palm oil and palm kernel oil alcoholy sis

Effect of various organic solvents on palm oil alcoholysis

Effect of various organic solvents on palm kernel oil alcoholysis

Alcoholysis reaction of palm oil a t optimum condition

Initial reaction rate of alcoholysis as a function of palm oil concentration

Initial reaction rate of alcoholysis as a function of palm kernel oil concentration

Initial reaction rate of palm oil alcoholysis as a function of oleyl alcohol concentration Initial reaction rate of palm kernel oil alcoholysis as a function of oleyl alcohol concentration

Lineweaver-Burk plot of alcoholysis as a function of palm oil concentration

Lineweaver-Burk plot of alcoholysis as a function of palm kernel oil concentration

Lineweaver-Burk plot of palm oil alcoholysis as a function of oleyl alcohol concentration

Lineweaver-Burk plot of palm kernel oil alcohol as a function of oleyl alcohol concentration

xxii

Response surface plot showing the effect of incubation time, temperature and their mutual effect on the synthesis of esters from palm oil alcoholysis

Response surface plot showing the effect of incubation time, temperature and their mutual effect on the synthesis of esters from palm kernel oil alcoholysis

Response surface plot showing the effect of molar ratio, temperature and their mutual effect on the synthesis of esters from palm oil alcoholysis

Response surface plot showing the effect of molar ratio, temperature and their mutual effect on the synthesis of esters from palm oil kernel alcoholysis

Response surface plot showing the effect of amount of enzyme, temperature and their mutual effect on the synthesis of esters of palm oil alcoholysis.

Response surface plot showing the effect of amount of enzyme, temperature and their mutual effect on the synthesis of esters of palm kernel oil alcoholysis.

Response surface plot showing the effect of molar ratio, time and their mutual effect on the synthesis of esters from palm oil alcoholysis

Response surface plot showing the effect of molar ratio, time and their mutual effect on the synthesis of esters from palm oil alcoholysis

Response surface plot showing the effect of amount of enzyme, time and their mutual effect on the synthesis of esters from palm oil alcoholysis

xxiii

Response surface plot showing the effect of amount of enzyme, time and their mutual effect on the synthesis of esters from palm kernel oil alcoholysis.

Response surface plot showing the effect of amount of enzyme, molar ratio and their mutual effect on the synthesis of esters from palm oil alcoholysis

Response surface plot showing the effect of amount of enzyme, molar ratio and their mutual effect on the synthesis of esters from palm oil alcoholysis

Contour plots of percentage yield of esters from alcoholysis of palm oil at molar ratio =1:1-1:5 and amount of enzyme = O.1g

Contour plots of percentage yield of esters from alcoholysis of palm oil at molar ratio =1:1-1:5 and amount of enzyme= 0.15 g

Contour plots of percentage yield of esters from alcoholysis of palm oil a t molar ratio = 1 : 1 - 1 :5 and amount of enzyme = 0.2 g

Contour plots of percentage yield of esters from alcoholysis of palm kernel oil at molar ratio =1: 1- 1:s and amount of enzyme = 0.1 g

Contour plots of percentage yield of esters from alcoholysis of palm kernel oil a t molar ratio = 1 : 1 - 1 :5 and amount of enzyme = 0.15 g

Contour plots of percentage yield of esters from palm kernel oil alcoholysis at molar ratio =1:1-1:5 and amount of enzyme = 0.2 g

Alcoholysis reaction of palm oils a t optimum condition by RSM

xxiv